Microfluidic device for cell separation and uses thereof

Abstract

The invention features methods for separating cells from a sample (e.g., separating fetal red blood cells from maternal blood). The method begins with the introduction of a sample including cells into one or more microfluidic channels. In one embodiment, the device includes at least two processing steps. For example, a mixture of cells is introduced into a microfluidic channel that selectively allows the passage of a desired type of cell, and the population of cells enriched in the desired type is then introduced into a second microfluidic channel that allows the passage of the desired cell to produce a population of cells further enriched in the desired type. The selection of cells is based on a property of the cells in the mixture, for example, size, shape, deformability, surface characteristics (e.g., cell surface receptors or antigens and membrane permeability), or intracellular properties (e.g., expression of a particular enzyme).

1 - 44 . (canceled) 
     
     
         45 . A microflow device comprising: a body having a randomized flow path which comprises an inlet, an outlet, and a microchannel arrangement extending between said inlet and outlet, wherein said microchannel arrangement is housed in a cavity in a surface of said body and comprises a plurality of transverse separator posts integral with a base surface of said cavity and projecting therefrom, wherein said posts are arranged in a random and irregular pattern; a closure plate having a top surface that is in contact with the surface of said body so that said cavity is closed; and a polymeric wrap encircling said body and said closure plate. 
     
     
         46 . The device of  claim 45 , wherein said body is molded from a flexible, optically transparent polymeric material. 
     
     
         47 . The device of  claim 46 , wherein said polymeric material is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate, polystyrene, and polyethylene teraphthalate. 
     
     
         48 . The device of  claim 45 , wherein said body is molded with a tab extending from one longitudinal end thereof. 
     
     
         49 . The device of  claim 45 , wherein the width of said closure plate is wider than the width of said body. 
     
     
         50 . The device of  claim 45 , further comprising a cap superimposed atop said body so that said encircling polymeric wrap sandwiches said body between said cap and said closure plate. 
     
     
         51 . The device of  claim 50 , wherein said cap has longitudinal sides that are beveled. 
     
     
         52 . The device of  claim 50 , wherein said cap has a width, at its widest dimension, equal to between about 75% and 100% of said body. 
     
     
         53 . The device of  claim 45 , wherein said polymeric wrap is a heatshrunken sleeve of polymeric material. 
     
     
         54 . The device of  claim 45 , wherein said polymeric wrap can be cut to allow separation of said body from said closure plate. 
     
     
         55 . The device of  claim 45 , wherein said inlet and said outlet comprise passageways having axes which are aligned at between 120° and 150° to said flat bottom surface of said body. 
     
     
         56 . The device of  claim 55 , wherein said axes lie in a vertical plane substantially perpendicular to said flat bottom surface of said body and are angularly aligned at between 80° and 100° to each other. 
     
     
         57 . The device of  claim 45 , wherein said inlet comprises a well capable of holding a liquid sample. 
     
     
         58 . The device of  claim 45 , wherein said posts are substantially perpendicular to the base surface of said cavity. 
     
     
         59 . The device of  claim 45 , wherein said posts extend to the top surface of said closure plate. 
     
     
         60 . The device of  claim 45 , wherein said closure plate is affixed to distal ends of said posts. 
     
     
         61 . The device of  claim 45 , wherein said posts have at least about 3 different cross sectional sizes. 
     
     
         62 . The device of  claim 45 , wherein said posts are between about 70 microns to about 130 microns in diameter. 
     
     
         63 . The device of  claim 45 , wherein the minimum separation between said posts is about 50 μM. 
     
     
         64 . The device of  claim 45 , wherein the total volume of said posts occupies about 15% to 25% of the total volume of said microchannel. 
     
     
         65 . The device of  claim 45 , wherein the randomized flow path provided by said posts disrupts stream-lined flow in said microchannel. 
     
     
         66 . The device of  claim 45 , wherein the randomized flow path provided by said posts prevents straight-line flow in said microchannel. 
     
     
         67 . The device of  claim 45 , wherein the surface of said microchannel is derivatized or coated to facilitate the attachment of a sequestering agent. 
     
     
         68 . The device of  claim 45 , wherein the surface of said microchannel is coated with a layer of hydrogel at least about 1 micron thick. 
     
     
         69 . The device of  claim 68 , wherein said hydrogel comprises an isocyanate-functional prepolymer comprising PEG, PPG, or a copolymer thereof. 
     
     
         70 . The device of  claim 45 , wherein a sequestering agent is directly or indirectly attached to the surface of said microchannel. 
     
     
         71 . The device of  claim 45 , wherein a sequestering agent is attached to the surface of said microchannel via a hydrophilic linker or a layer of hydrogel. 
     
     
         72 . The device of  claim 45 , wherein the surface of said microchannel is coated with a sequestering agent selected from the group consisting of antibody, antigen, receptor, ligand, oligonucleotide, and peptide. 
     
     
         73 . The device of  claim 45 , wherein said closure plate is optically transparent. 
     
     
         74 . A method of capturing a target biomolecule comprising causing a sample containing a target biomolecule to flow through said microchannel of the device of  claim 45 , wherein the surface of said microchannel is coated with a sequestering agent capable of binding to said target biomolecule. 
     
     
         75 . The method of  claim 74 , wherein the sample flows through said microchannel at a rate of about 0.1 mm to about 2 mm per second. 
     
     
         76 . The method of  claim 74 , wherein the sample flows through said microchannel at a rate of about 0.2 mm to about 1 mm per second. 
     
     
         77 . A method of detecting a target biomolecule in a sample comprising: causing a sample to flow through said microchannel of the device of  claim 45 , wherein the surface of said microchannel is coated with a sequestering agent capable of binding to the target biomolecule; and detecting the target biomolecule on the surface of said microchannel. 
     
     
         78 . The method of  claim 77 , wherein the target molecule is a cell. 
     
     
         79 . The method of  claim 78 , wherein the cell is a fetal cell. 
     
     
         80 . The method of  claim 77 , wherein the cell is a cancer cell or a tumor cell. 
     
     
         81 . The method of  claim 77 , wherein said sample comprises blood or cervical mucous. 
     
     
         82 . The method of  claim 77 , wherein the target molecule is a virus, polynucleotide, protein, or carbohydrate. 
     
     
         83 . The method of  claim 77 , wherein the sample flows through said microchannel at a rate of about 0.1 mm to about 2 mm per second. 
     
     
         84 . The method of  claim 77 , wherein the sample flows through said microchannel at a rate of about 0.2 mm to about 1 mm per second. 
     
     
         85 . The device of  claim 45 , wherein the spacing between said posts is variable. 
     
     
         86 . The device of  claim 45 , wherein the microchannel arrangement comprises a collection region, and wherein said posts are arranged in a random and irregular pattern across the entire width of said collection region. 
     
     
         87 . A microflow apparatus comprising: a body having a randomized flow path which comprises an inlet, an outlet, and a microchannel arrangement extending between said inlet and outlet, wherein said microchannel arrangement includes a plurality of transverse separator posts being integral with a base surface of said microchannel and projecting thereform, wherein said posts are arranged in a pattern capable of providing said randomized flow path, and wherein said posts contact both the top and bottom of said microchannel, wherein said apparatus is held together by external means. 
     
     
         88 . The apparatus of  claim 87  wherein said body is molded from a polymeric material. 
     
     
         89 . The apparatus of  claim 87 , wherein said polymeric material is selected from the group consisting of poly(dimethylsiloxane), poly(methylmethacrylate) (PMMA), polycarbonate, polystyrene, and polyethylene. 
     
     
         90 . The apparatus of  claim 87  wherein said body is molded with a tab extending from one longitudinal end thereof. 
     
     
         91 . The apparatus of  claim 87  wherein the width of said top is wider than said body. 
     
     
         92 . The apparatus of  claim 87  wherein said inlet is configured to accept fluid sample from a sample reservoir. 
     
     
         93 . The apparatus of  claim 87  wherein said posts are substantially perpendicular to the base surface of the body. 
     
     
         94 . The apparatus of  claim 87  wherein said posts extend to the top of said body. 
     
     
         95 . The apparatus of  claim 87  wherein said top is affixed to a first end of said posts. 
     
     
         96 . The apparatus of  claim 87  wherein the posts comprise a variety of cross sectional sizes. 
     
     
         97 . The apparatus of  claim 87  wherein the posts have a diameter that is at least 50 microns and less than 250 microns. 
     
     
         98 . The apparatus of  claim 87  wherein spacing between posts is 50 microns. 
     
     
         99 . The apparatus of  claim 87  wherein said posts occupy a relatively small percentage of the total volume of said body. 
     
     
         100 . The apparatus of  claim 87 , wherein posts are configured to shift cells off their streamlines. 
     
     
         101 . The apparatus of  claim 87 , wherein posts are configured to shift cells off their streamlines. 
     
     
         102 . The apparatus of  claim 87 , wherein surface of said body is derivatized with binding moieties. 
     
     
         103 . The apparatus of  claim 87 , wherein apparatus comprises loosely cross-linked hydrogel. 
     
     
         104 . The apparatus of  claim 103  wherein said apparatus comprises PEG. 
     
     
         105 . The apparatus of  claim 87 , wherein a binding moiety is in contact with said microchannel. 
     
     
         106 . The apparatus of  claim 87  wherein said binding moiety is contacted with a layer of a charge polymer. 
     
     
         107 . The apparatus of  claim 87 , wherein surface of said microchannel is coated with a binding moiety selected from the group consisting of antibody, a charged polymer, a molecule that binds to a cell surface receptor, an oligo- or polypeptide, a viral or bacterial protein, a nucleic acid, and a carbohydrate. 
     
     
         108 . The apparatus of  claim 87 , wherein said apparatus is optically transparent. 
     
     
         109 . A method for capturing a target cell comprising causing a sample containing a rare cell to flow through said microchannel of  claim 87 , wherein surface of said microchannel is coated with a binding moiety capable of binding said rare cell. 
     
     
         110 . The method of  claim 109  wherein the sample flows through said microchannel at a rate of at most 15 ul/sec or 0.25 to 1 mL/h. 
     
     
         111 . A method of detecting a biomolecule in a sample comprising: causing a sample to flow through said microchannel of the apparatus of  claim 87 , wherein surface of said microchannel is coated with a binding moiety capable of binding the biomolecule, and detecting the biomolecule on the surface of said microchannel. 
     
     
         112 . The method of  claim 111 , wherein the biomolecule is a cell. 
     
     
         113 . The method of  claim 111 , wherein the biomolecule rare cell is a fetal cell. 
     
     
         114 . The method of  claim 111 , wherein the biomolecule is a cancer cell or a tumor cell. 
     
     
         115 . The method of  claim 111 , wherein the sample is a blood sample. 
     
     
         116 . The method of  claim 111 , wherein the biomolecule is a membrane fragments or proteins. 
     
     
         117 . The method of  claim 111 , wherein the sample flows through said microchannel at a rate of at most 15 ul/sec or 0.25 to 1 mL/h. 
     
     
         118 . The device of  claim 87 , wherein the spacing between the posts is variable. 
     
     
         119 . The device of  claim 87 , wherein the microchannel comprises a collection of regions.